﻿Reassortant
Avian Influenza
Virus (H5N1)
in Poultry,
Nigeria, 2007
Isabella Monne,*1
Tony M. Joannis,1
Alice Fusaro,* Paola De Benedictis,*
Lami H. Lombin, Husseini Ularamu,
Anthony Egbuji, Poman Solomon,
Giovanni Cattoli,* Ilaria Capua,* and Tim U. Obi
Genetic characterization of a selection of influenza vi-
rus (H5N1) samples, circulating in 8 Nigerian states over
a 39-day period in early 2007, indicates that a new reas-
sortant strain is present in 7 of the 8 states. Our study re-
ports an entirely different influenza virus (H5N1) reassortant
becoming predominant and widespread in poultry.
Since its emergence in 2006 in Africa, avian influenza vi-
ruses of the H5N1 subtype have spread rapidly to poul-
try farms in several African countries. In February 2006,
Kaduna State in Nigeria was the first of 36 states to report
infection of poultry with highly pathogenic avian influenza
virus (H5N1). Currently, infection has spread to 22 of the
36 Nigerian states and to the Federal Capital Territory. In
February 2007, 1 case of avian influenza was reported in a
woman from the southern state of Lagos. Thus, the exten-
sive circulation of influenza virus (H5N1) in Nigeria raises
concerns about human and animal health issues. A previ-
ous study indicated that 2 sublineages (EMA1 and EMA2)
were cocirculating in Nigeria in 2006 (1); however, 3 sub-
lineages were identified in a more recent study (2), namely
sublineage A (corresponding to EMA2) and sublineages B
and C (corresponding to EMA1). The 2007 study by Salz-
berg et al. also identified a virus showing a 4:4 reassort-
ment between genes of sublineages EMA1 and EMA2 (1).
The aim of our study is to provide additional information
on the genetic characteristics of isolates that were circulat-
ing in Nigeria in early 2007.
The Study
Twelve representative influenza virus (H5N1) samples
from different Nigerian outbreaks were selected (Table 1)
by taking into account the geographic origin and the date of
isolation. We then characterized these viruses by sequenc-
ing the entire genome.
Samples were processed for virus isolation, subtyp-
ing, and pathotyping (3,4). The amplification of the 8 viral
gene segments was carried out with reverse transcription
(RT)­PCR by using gene-specific primers (available upon
request). PCR products were sequenced in a 3100 Avant
Genetic Analyzer (Applied Biosystems, Foster City, CA,
USA). Phylogenetic analysis was performed by using the
neighbor-joining method as implemented in the MEGA 3
program, ClustalW software (www.ebi.ac.uk/Tools/
clustalw2/index.html) (5). GenBank accession nos. for the
8-gene segments of the 12 Nigerian strains are EU148356
to EU148451.
As expected, all Nigerian isolates were closely related
to the viruses that have been circulating in birds through-
out Europe, Russia, Africa, and the Middle East since late
2005. According to the unified nomenclature system for
highly pathogenic influenza virus (H5N1), these isolates
belong to clade 2.2 (6).
Phylogenetic analysis of all 8 gene segments of the
recent Nigerian strains showed that 10 of these strains, la-
beled EMA1/EMA2-2:6 reassortant 2007 (EMA1/EMA2-
2:6-R07) (Table 1), have the same genotype. In particular,
the genetic comparison of the hemagglutinin (HA) and non-
structural (NS) genes of EMA1/EMA2-2:6-R07 shows that
they are derived from viruses of the EMA1 sublineage (2)
and have the highest similarity with the first Nigerian strain
isolated, A/chicken/Nigeria/641/2006 (homology ranged
between 99.3% and 99.7%). We observed different topol-
ogy for the remaining gene segments (neuraminidase, nu-
cleoprotein [NP], matrix, heterotrimeric polymerase com-
plex [PA, PB1, and PB2]). Phylogenetic analysis showed
that the nucleotide sequences of these genes fall into EMA2
sublineage; the highest homology was observed with re-
spective gene segments of the Nigerian strains isolated in
2006, which belong to EMA2 (homology ranged between
99.4% and 99.7%). The separation of the gene segments
into 2 clusters (Figures 1, 2) is evidence of reassortment
(7). The genetic pattern of EMA1/EMA2-2:6-R07 virus is
distinct from that of A/chicken/Nigeria/1047-62/2006 vi-
rus isolated in June, 2006 in Taraba State and recognized
previously as an EMA1/EMA2-4:4 reassortant virus (2)
(Table 2). The remaining 2 viruses were not reassortants.
They were detected in Sokoto State and belong to sublin-
eage EMA2.
Sequence analysis showed that the receptor binding
site of all of the Nigerian viruses under study retains amino
acid residues (Gln 222 and Gly 224). These residues pref-
erentially recognize receptors with saccharides terminating
in sialic acid-2-3-galactose (SA2-3Gal), specific for avi-
an species (8). Mutations that are related to neuraminidase
Emerging Infectious Diseases · www.cdc.gov/eid · Vol. 14, No. 4, April 2008 637
*Viale dell'Università, Legnaro, Padova, Italy; National Veterinary
Research Institute, Plateau State, Vom, Nigeria; and University of
Ibadan, Oya State, Ibadan, Nigeria 1
These authors contributed equally to this article.
inhibitor and adamantanes resistance were not detected in
any of the 12 isolates (9). All 12 Nigerian viruses possess
the PB2 627K mutation associated with increased virulence
of influenza virus (H5N1) in mice (10). Two other host spe-
cific mutations were observed for NP and PA genes. In par-
ticular, the NP gene contains a human specific amino acid
signature at position 33 (V33I) (11) in all selected isolates.
A human specific amino acid signature was also detected
at position 100 (V100A) of the PA gene of A/chicken/
Nigeria/1071-3/2007 and A/chicken/Nigeria/1071-7/2007
strains (12).
Conclusions
The results of our study show that 10 of 12 strains ob-
tained over a 39-day period were EMA1/EMA2-2:6-R07
reassortant viruses, and that these were circulating in at
least 7 Nigerian states. This appears to be the only report
of a reassortant virus generated by H5N1 viruses belong-
ing to the 2.2 clade spreading extensively in poultry. Thus,
the viruses circulating in 2007 in Nigeria differ from the
original sublineage prototypes introduced during 2006. Our
findings also suggest that an influenza virus (H5N1) with
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638 Emerging Infectious Diseases · www.cdc.gov/eid · Vol. 14, No. 4, April 2008
Table 1. List of influenza virus (H5N1) samples analyzed in poultry, Nigeria, 2007
Virus Group State of isolation Date of isolation
A/chicken/Nigeria/1071-1/2007 EMA1/EMA2-2:6-R07 Plateau Jan 2
A/chicken/Nigeria/1071-3/2007 EMA2 Sokoto Jan 5
A/chicken/Nigeria/1071-4/2007 EMA1/EMA2-2:6-R07 Borno Jan 5
A/chicken/Nigeria/1071-5/2007 EMA1/EMA2-2:6­R07 Plateau Jan 6
A/chicken/Nigeria/1071-7/2007 EMA2 Sokoto Jan 10
A/chicken/Nigeria/1071-9/2007 EMA1/EMA2-2:6-R07 Bauchi Jan 12
A/chicken/Nigeria/1071-10/2007 EMA1/EMA2-2:6-R07 Anambra Jan 13
A/chicken/Nigeria/1071-15/2007 EMA1/EMA2-2:6-R07 Kaduna Jan 23
A/chicken/Nigeria/1071-22/2007 EMA1/EMA2-2:6-R07 Kano Jan 31
A/duck/Nigeria/1071-23/2007 EMA1/EMA2-2:6-R07 Borno Feb 1
A/chicken/Nigeria/1071-29/2007 EMA1/EMA2-2:6-R07 Lagos Feb 9
A/chicken/Nigeria/1071-30/2007 EMA1/EMA2-2:6-R07 Kaduna Feb 10
Figure 1. Phylogenetic tree for the hemagglutinin gene of influenza
viruses constructed by neighbor-joining method. Sequences
obtained in this study were labeled with a circle (EMA1/EMA2-2:6-
R07 group) and triangle (EMA2 group). The remaining sequences
can be found in GenBank. The numbers at each branch point
represent bootstrap values, and they were determined by bootstrap
analysis by using 1,000 replications. Scale bar = 0.01 nucleotide
substitutions/site.
Figure 2. Phylogenetic tree for nucleoprotein gene of influenza
viruses constructed by neighbor-joining method. Sequences
obtained in this study were labeled with a circle (EMA1/EMA2-2:6-
R07 group) and triangle (EMA2 group). The remaining sequences
can be found in GenBank. The numbers at each branch point
represent bootstrap values, and they were determined by bootstrap
analysis by using 1,000 replications. Scale bar = 0.01 nucleotide
substitutions/site.
Reassortant Influenza (H5N1) in Poultry, Nigeria
new genetic characteristics has emerged in <7 months and
is widespread in Nigeria.
The emergence of at least 2 reassortant viruses in
Nigeria shows that co-infection with viruses of different
sublineages has occurred presumably in poultry. This evi-
dence is most likely a result of poor biosecurity measures
implemented by the poultry industry, particularly the live-
bird market system, which is known to facilitate mingling
of infected birds. In nonindustrialized countries, live bird
market systems sometimes allow birds of different spe-
cies and of unknown health status to share limited space,
often the same cage. Birds in the incubation stage or
breeds that show a reduced clinical susceptibility may not
appear overtly ill and therefore, may be traded in live bird
market systems. The movement of infected birds across
neighboring regions could explain the genetic relatedness
found between influenza virus (H5N1) isolates obtained
from 7 Nigerian states. The predominance of a reas-
sortant virus in Nigeria mimicks the previously reported
predominance of the Z genotype virus in Asia, although
this genotype is believed to contain internal genes origi-
nating from noninfluenza viruses (13). The introduction
of influenza virus (H5N1) of different clusters in 2005 in
Vietnam, also resulted in the emergence of a reassortant
strain. Unlike the Nigerian situation described here, the
Vietnamese reassortant influenza virus (H5N1) did not
become predominate in Vietnam (2).
Currently, the genetic characteristics of the human Ni-
gerian isolate are unavailable. Given that human infection
occurs after direct contact with infected poultry, studies
need to be performed to establish whether the predominant
avian influenza virus (EMA1/EMA2-2:6-R07) has also
been responsible for human infection. Amino acid muta-
tions towards increased binding to human receptors, name-
ly N182K, Q192R, Q226L, and G228S substitutions in the
HA gene (14,15), do not seem to have occurred in Nigerian
influenza (H5N1) strains to date. However, 2 molecular
changes, the 33 NP valine to isoleucine substitution, which
is typical of human influenza viruses (11), and the 627 PB2
glutamic acid to lysine mutation, which increases virulence
in the mammalian host, have been detected in all analyzed
strains (10).
The results of our study suggest that depopulation and
biosecurity measures adopted in Nigeria are not sufficient
to prevent the spread of the virus and should be improved.
Poultry farmers and smallholder poultry producers (those
producing a limited number of poultry) must be educated
on appropriate control measures for avian influenza. In ad-
dition, isolation and genetic characterization of African in-
fluenza virus (H5N1) isolates in a transparent environment,
should be promoted and supported so that more information
can be gathered on the evolution of this virus in Africa.
Acknowledgments
We thank FAO/Animal Health Service of the Animal Pro-
duction and Health Division of FAO for supporting sequence
analysis of strains and facilitating samples submission and Katha-
rine Sturm-Ramirez for her assistance.
This study was possible through contacts established in the
framework of the Technical Cooperation Projects funded by the
Food and Agriculture Organization of the United Nations (FAO).
The work was also funded and supported by the EU Projects:
Generation of information and tools to support the management
of the avian influenza crisis in poultry (FLUAID) and Training
and technology transfer of avian influenza diagnostics and disease
management skills (FLUTRAIN).
Dr Monne is a specialist in veterinary public health and food
safety of animal products. She is currently working at the Labo-
ratory of Molecular Biology, which is included in the National
and World Organization for Animal Health (OIE)/FAO Reference
Laboratory for Newcastle disease and avian influenza. Her main
fields of research are the development and application of rapid
and novel diagnostic methods for infectious diseases of animals
Emerging Infectious Diseases · www.cdc.gov/eid · Vol. 14, No. 4, April 2008 639
Table 2. Clustering of the gene segments of influenza strains in poultry, Nigeria, 2007*
Virus HA NA NS MA PB2 PB1 PA NP
A/chicken/Nigeria/641/2006 EMA1 EMA1 EMA1 EMA1 EMA1 EMA1 EMA1 EMA1
A/chicken/Nigeria/1047-62/2006 EMA1 EMA2 EMA1 EMA2 EMA2 EMA1 EMA2 EMA1
A/chicken/1071-1/Nigeria/2007
A/chicken/1071-4/Nigeria/2007
A/chicken/1071-5/Nigeria/2007
A/chicken/1071-9/Nigeria/2007
A/chicken/1071-10/Nigeria/2007
A/chicken/1071-15/Nigeria/2007
A/chicken/1071-22/Nigeria/2007
A/duck/1071-23/Nigeria/2007
A/chicken/1071-29/Nigeria/2007
A/chicken/1071-30/Nigeria/2007
EMA1 EMA2 EMA1 EMA2 EMA2 EMA2 EMA2 EMA2
A/chicken/1071-3/Nigeria/2007
A/chicken/1071-7/Nigeria/2007
EMA2 EMA2 EMA2 EMA2 EMA2 EMA2 EMA2 EMA2
*HA, hemagglutinin; NA, neuraminidase; NS, nonstructural; MA; matrix; PB2; polymerase B2; NP, nucleoprotein.
These strains were analyzed in a previous study (2).
and molecular epidemiology of avian viruses, particularly avian
influenza viruses with human health implications.
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Address for correspondence: Ilaria Capua, Viale dell'Università 10,
35020, Legnaro, Padova, Italy; email: icapua@izsvenezie.it
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